A FRET-Based Assay for the Identification of PCNA Inhibitors

Abstract

Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA–p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA–p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA–p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S–p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.

Identification of a potent PCNA‐p15‐interaction inhibitor by autodisplay‐based peptide library screening

“…Allosteric inhibitors would be a new class of PCNA interaction inhibitors allowing the selective regulation of the target function and facilitating a better estimation of the inhibitory effect (Ni et al., 2019 ). Allosteric stabilizers might be used for the therapy of Ataxia‐telangiectasia‐like disorder type 2, in which the stability of PCNA is dramatically impaired (Baple et al., 2014 ; Hardebeck et al., 2023 ).…”

“…Purification of mScarlet‐I‐PCNA was conducted according to the protocol of (Hardebeck et al., 2023 ). In short, the expression of mScarlet‐I‐PCNA was induced at an OD 578nm of 0.6 by the addition of 1 mM isopropyl‐β‐D‐thiogalactopyranoside (IPTG) and incubation of the cells at 23°C for 16 h. mScarlet‐I‐PCNA was purified from the bacterial lysate by immobilized metal ion affinity chromatography through the N‐terminal integrated His 6 epitope.…”

Application of an alchemical free energy method for the prediction of thermostable DuraPETase variants

“…A convenient way to detect such limitations during MD sampling could be the usage of closed thermodynamic cycles with relative free energy differences at their edges. If the sum of all relative free energy changes deviates from zero, insufficient sampling for one or multiple of the mutations could be an issue (Gapsys et al 2015a ; Hardebeck et al 2023 ). Both approaches mentioned above, the use of closed thermodynamic cycle and increasing the number of independent replicas, come however with a substantial increase in computational effort and need to be considered carefully.…”

“…The codon-optimized sequence of the DuraPETase gene is given in Sequence S1. It was inserted into the plasmid p15-mNeonGreen (Hardebeck et al 2023 ) by restriction and ligation to create the plasmid pDuraPETase for intracellular expression of the original DuraPETase under control of a T7 promotor. Point mutations for the generation of DuraPETase variants were introduced by PCR via In-Fusion cloning (Takara Bio, Kusatsu, Japan).…”

“…This approach has been shown to give good prediction accuracy in retrospective studies, in which literature values were used to verify the validity of the predictions. Recently, the approach was also used prospectively to guide the functional analysis of a disease conferring mutation in the proliferating cell nuclear antigen followed by experimental validation (Hardebeck et al 2023 ). Prospective, larger scale studies employing this method for the prediction of more thermostable enzyme variants, followed by experimental validation, are however scarce.…”